1. Field of the Invention
This invention relates to flow cytometry and, more particularly, to an improved flow cytometry apparatus and method involving a novel time delayed integration of a charge coupled device in the detection apparatus for enhancing the signal-to-noise ratio.
2. The Prior Art
Cytometry is the measurement of the physical and/or chemical characteristics of biological cells. Since the technology of my novel discovery as disclosed and claimed herein can be adapted to other systems such as viruses and molecules as well as biological cells, I have adopted the term "cytometry" to refer to the detection of all of these species which, for the sake of convenience are referred to herein as "target particle(s)." Flow cytometry, as the name implies, involves the delivery of a flowing stream containing a sample having target particles therein to the detection region of a flow cytometer. The target particles in the detection region are irradiated using a laser to create an illumination phenomena by the target particles. The appropriate optics and detection electronics measure the light absorption; scattering; fluorescence; and/or spectral properties of the target particles in the sample, or alternatively, a fluorescent label affixed to the target particles. The laser is typically a gas laser (such as an Argon or a Helium Neon Laser) or even a diode laser. In the case of fluorescence, each target particle produces a burst of fluorescence photons as it passes through the illumination region. Differentiation of the fluorescence from the illumination or excitation light can be accomplished with a filter or a combination of filters. Detection of the fluorescence is accomplished using a photomultiplier tube or a photodiode. Another technique relies on light scattering of photons in the illumination beam by the target particle. The target particle is identified by its light scattering as a function of the angle of scattering which is a function of its size and shape as well as the wavelength of the scattered photons.
The successful detection and identification of a single target particle depends upon several factors. First, the laser power must be sufficient to generate a large enough number of fluorescence (or, alternatively, scattering) photons during the brief passage of the target particle through the irradiation region. Specifically, it is essential that a sufficient number of photons are generated so that the fluorescent burst from the target particle can be reliably differentiated from random fluctuations in the number of background photons. Second, it is important to minimize these unwanted background photons. These unwanted background photons arise from scattering or from fluorescence emitted by the carrier liquid of the sample or impurities in the liquid, as well as from the apparatus itself, such as the walls of the capillary through which the flow stream passes.
Flow cytometry inherently creates competition between a desirable high flow rate for a reasonable sample throughput in order to provide rapid detection of target particles and high detectivity. High detectivity is predicated upon having a fluorescing (or scattering) target particle in the detection beam long enough to provide a high signal-to-noise detection along with an optical design that will minimize background photons from unwanted scattering and unwanted fluorescence, both of which can dominate system noise. Optimally, a flow cytometer will have a small excitation beam, a high flow rate, and detection optics and associated electronics that allow for the collection of sufficient photons from the target particle to provide high signal-to-noise detection.
Various flow cytometry devices are described in available references. See, for example Flow Cytometry: First Principles, A. L. Givan (1992). Other references include Sage et al. (U.S. Pat. No. 4,660,971); Wu et al. (U.S. Pat. No. 4,661,913); Yamamoto et al. (U.S. Pat. No. 4,786,165); Ohki et al. (U.S. Pat. No. 5,007,732); and Hirako (U.S. Pat. No. 5,135,302). Each of these references also contain references to other relevant publications.
As discussed previously, the competing requirements imposed on flow cytometry create an inherent conflict between high sample throughput and high detectivity that has proven most difficult to overcome resulting in limitations in applications for flow cytometry. In view of the foregoing, it would be an advancement in the art to provide improvements in flow cytometry. It would be another advancement in the art to provide a charge coupled device (CCD) as the detector with the image from the target particle being shifted across the CCD at the same rate that the CCD charge is being shifted across the CCD. It would be an even further advancement in the art to provide a flow cytometer with a time delayed integration (TDI) system to significantly alter the signal-to-noise ratio in favor of detection. Such a novel apparatus and method is disclosed and claimed herein.